Isolation And Characterization Of Terpene Synthases And Corresponding Promoters In Artemisia Annua L.

Terpenoids,consisting of isoprenoid structural units?C₅?,are the largest family of natural compounds.They are important components of plant secondary metabolites.Varied terpenoids synthesized by plants are related to defense against biotic and abiotic stresses.Moreover,terpenoids possess critical economic values in that they can be used as fragrance,flavor,medicine and pesticide.Terpene synthases usually catalyze the first key reaction of terpene biosynthetic pathway.They are regulated by diverse factors including transcription factors,phytohormones,stresses,constituting a complicated net of plant secondary metabolism.Artemisia annua L.is an annual herb,whose metabolite of artemisinin plays an important role in treating malaria.Besides,the essential oil of A.annua is rich in monoterpenes and sesquiterpenes with distinct functions,such as 1,8-cineole,caryophyllene and artemisia ketone.Key structural enzymes in the artemisinin biosynthetic pathway have been discovered,including ADS,CYP71AV1,DBR2 and ALDH1.However,lots of other terpene synthases remain to be cloned and characterized in order to get in-depth knowledge of the functions and structures of terpenoids.Two putative terpene synthase genes,AaTPS7 and AaTPS8,were cloned from the A.annua trichome transcriptome library.AaTPS7 is 1863 bp long encoding a protein of 620 amino acids.AaTPS8 is 1683 bp and encodes a protein of 560 amino acids.It is indicated that AaTPS7 and AaTPS8 are highly homologous with monoterpenes and sesquiterpenes respectively of different species via bioinformatics.Enzyme assay showed that AaTPS8 was a sesquiterpene synthase.It catalyzed FPP into three products among which intermedeol was the main product.Although no product was detected of AaTPS7's catalysis assay via GC-MS analysis,the results of multi-sequence alignment and phylogenetic analysis indicated that it was a monoterpene synthase.Expression profile of AaTPS7 and AaTPS8 at varied tissues and different positions of leaves were analyzed by RT-qPCR and the results showed a relatively higher expression in young leaf,stem or bud.The promoter regions of AaTPS7 and AaTPS8 were isolated respectively by genomic walking method.proTPS7 is 1167 bp long and proTPS8 is 1629bp long.Several cis-acting elements were discovered via bioinformatics including WRKY binding site W-box,MYB binding site MBS,MeJA responsive element CGTCA-motif and ABA responsive element ABRE.It is indicated that the two promoters may be regulated by various factors such as transcription factors,wound,etc.Then proTPS7 and proTPS8 were fused to GUS reporter gene and transformed into A.annua.GUS staining assay showed that they induced non-glandular trichome specific expression of GUS,while no staining was observed in glandular trichomes.As glandular trichomes are the places where artemisinin is synthesized and stored,it is indicated that the two genes are not involved in artemisinin biosynthetic pathway.After treated by MeJA and ABA,GUS expression in transgenic A.annua was improved 3hours later,indicating that proTPS7 and proTPS8 were hormone responsive.Constitutive promoters are widely used in genetic engineering but not ideal tools enough.Trichome specific promoters may serve as options replacing constitutive promoters for precise regulation in genetic and metabolic engineering.In this study,AaTPS7 and AaTPS8 as well as their corresponding promoters were isolated and characterized,providing possibilities to get better knowledge of secondary metabolism and the regulation of terpenoids biosynthetic pathway to increase the content of target compounds.